Project Summary Human cytomegalovirus (HCMV) retinitis initially resulted in vision loss and blindness in ~30% of HIV/AIDS patients before development of combination antiretroviral therapy (cART), yet this slowly progressive retinal disease remains a significant ophthalmologic problem in HIV/AIDS patients worldwide who do not respond to cART or who discontinue therapy. Chronic infection with HCMV is a risk factor for the progression of age- related macular degeneration (AMD). A critical barrier to progress in treating and preventing HCMV retinitis is limited understanding of the mechanism of uninfected bystander cell death and the lack of a preventative strategy. Our goal is to determine the role of receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) in innate immune responses and the death of uninfected bystander retinal cells during CMV infection. Our central hypothesis is that RIP1/RIP3 play critical roles in the death of uninfected bystander cells via AIF-mediated, caspase 3-independent apoptosis/necroptosis. Our objectives are to use an in vivo mouse model of CMV retinitis, an in vivo mouse model of choroidal MCMV infection and an in vitro organotypic retinal culture model to 1) demonstrate how RIP kinases promote death of uninfected bystander retinal cells either by directly activating necroptosis/apoptosis pathways or indirectly via apoptosis inducers downstream to activation of caspase 1 or NFkB; 2) identify the mechanism by which photoreceptor cell death is induced by RIP1/RIP3 activation and inflammation subsequent to choroidal MCMV infection and 3) begin development of a strategy to prevent cell death. Our expected outcomes include 1) activation of RIP1 and RIP3, which is negatively regulated by Bax and caspase 12, plays a central role in death of retinal neurons, especially photoreceptors, via AIF-mediated, caspase 3-independent apoptosis/necroptosis; and 2) specific inhibitors of RIP kinases can greatly reduce death of uninfected bystander retinal cells. The impact of our project includes 1) providing new information about the mechanism of CMV retinitis, 2) furthering our understanding of the complex interaction of cell death pathways which govern neuronal cell survival; 3) establishing a basis for development of pharmacological agents which prevent or reduce retinal neuron death and tissue damage in human patients with CMV retinitis or with other retinal diseases in which pathogenesis depends on or intersects with some of the same molecular pathways; and 4) increasing our understanding of the pathogenesis of AMD in human patients. Aim 1 will test the hypothesis that activation of RIP3 induces AIF-mediated, caspase 3- independent apoptosis/necroptosis directly or indirectly via inflammasome and NF?B downstream effectors. Aim 2 will test the hypothesis that Bax inhibits activation of RIP1 and subsequent AIF-mediated, caspase 3- independent apoptosis/necroptosis via promotion of caspase 12 activation. Aim 3 will test the hypothesis that inhibition of RIP kinases relieves photoreceptor death following choroidal MCMV infection.